Hepatitis C virus (HCV) is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma in the US and in many other parts of the world. Currently available therapy is not effective in many patients. Drug and alcohol abusers are especially at risk. A high percentage (up to -40%) of people with alcoholic liver disease show evidence of active infection by HCV. The prognosis for these people is significantly worse than for infected people without alcohol abuse, in that they show more hepatic dysfunction, increased hepatic pathology, and accelerated rates of liver fibrosis. Many studies also show increased levels of viral replication and hepatocellular carcinoma and lower response rate to therapeutic intervention. Therefore, there is an urgent need for novel drugs to block HCV replication in infected people who abuse alcohol. One approach to finding new drug targets is to look for specific virus-host interactions that are necessary for the viral life cycle. We are concentrating on the relationship between HCV proteins and lipid droplets, since hepatic steatosis is common in both alcoholic liver disease and chronic hepatitis C. HCV core protein is known to localize, in part, to the surface of lipid droplets. Another group has recently found that the NS5A protein is also localized to lipid droplets. This finding has been independently confirmed in our laboratory. We have also mapped a region of NS5A protein that can mediate the localization of heterelogous proteins to lipid droplets, and found a cellular lipid droplet protein that binds to this region of NS5A protein. The proposed experiments will test the hypothesis that this cellular protein mediates the localization of NS5A protein to lipid droplets, and that the binding of NS5A protein to this protein and lipid droplets is necessary for high-level HCV RNA replication. We will also set up a rapid screening system to look for small molecules that can disrupt the interaction between NS5A protein and the cellular protein. It is anticipated that these interdisciplinary experiments will lead to the discovery of novel therapeutic agents for blocking HCV replication, especially for people with steatosis.